Sustained availability of efficacious drugs is essential for worldwide efforts to eradicate malaria. The emergence and spread of drug resistance to current antimalarial therapies remains a pressing concern with reports of artemisinin-based treatment failures escalating the need for novel antimalarial chemotherapies. Thus the discovery of novel druggable targets and pathways, including those that are critical for multiple life stages, is a major challenge for the development of next-generation therapeutics. Using an integrated chemogenomic approach, we have previously identified the cytoplasmic prolyl tRNA synthetase in Plasmodium falciparum (PfcPRS) as the long-sought biochemical target of halofuginone. Furthermore, we uncovered an unprecedented mechanism of drug-tolerance to halofuginone in the parasite by modulation of proline homeostasis. However, small molecule inhibtors that bind PfcPRS in a mode distinct from halofuginone are active, not cross-resistant and elicit a distinct biological response. In this proposal, we seek to establish the molecular basis for this differential response and identify lead compounds that will allow us to further mechanistically probe the consequences of PfcPRS inhibition. We bring an integrated approach combining our expertise in molecular parasitology, genetics, metabolomics, structural biology, and rational inhibitor design to probe these aspects of aminoacyl tRNA synthetase biology and inhibition in the parasite. We will explore the inhibitor class dependent downstream signaling of PRS inhibition, evaluating the role of tRNA signaling and inhibition mode. Specifically we will mechanistically and functionally characterize the molecular consequences of PRS inhibition depending on the interference of the small molecule inhibitor with tRNA binding and develop new PfcPRS inhibitors that allow us to probe the relevance of inhibition of the tRNA binding pocket and demonstrate improved selectivity